TY - JOUR
T1 - A lipoprotein allosterically activates the CwlD amidase during Clostridioides difficile spore formation
AU - Feliciano, Carolina Alves
AU - Eckenroth, Brian E.
AU - Diaz, Oscar R.
AU - Doublié, Sylvie
AU - Shen, Aimee
N1 - Funding Information:
We would like to thank B. Jackson at the Dartmouth Trace Element Analysis Core for help with the elemental analyses; N. Minton (U. Nottingham) for generously providing us with access to the 630?erm?pyrE strain and pMTL-YN1C and pMTL-YN3 plasmids for allele-coupled exchange (ACE).
Publisher Copyright:
© 2021 Alves Feliciano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2021/9/27
Y1 - 2021/9/27
N2 - Spore-forming pathogens like Clostridioides difficile depend on germination to initiate infection. During gemination, spores must degrade their cortex layer, which is a thick, protective layer of modified peptidoglycan. Cortex degradation depends on the presence of the spore-specific peptidoglycan modification, muramic-@-lactam (MAL), which is specifically recognized by cortex lytic enzymes. In C. difficile, MAL production depends on the CwlD amidase and its binding partner, the GerS lipoprotein. To gain insight into how GerS regulates CwlD activity, we solved the crystal structure of the CwlD:GerS complex. In this structure, a GerS homodimer is bound to two CwlD monomers such that the CwlD active sites are exposed. Although CwlD structurally resembles amidase_3 family members, we found that CwlD does not bind Zn2+ stably on its own, unlike previously characterized amidase_3 enzymes. Instead, GerS binding to CwlD promotes CwlD binding to Zn2+, which is required for its catalytic mechanism. Thus, in determining the first structure of an amidase bound to its regulator, we reveal stabilization of Zn2+ co-factor binding as a novel mechanism for regulating bacterial amidase activity. Our results further suggest that allosteric regulation by binding partners may be a more widespread mode for regulating bacterial amidase activity than previously thought.
AB - Spore-forming pathogens like Clostridioides difficile depend on germination to initiate infection. During gemination, spores must degrade their cortex layer, which is a thick, protective layer of modified peptidoglycan. Cortex degradation depends on the presence of the spore-specific peptidoglycan modification, muramic-@-lactam (MAL), which is specifically recognized by cortex lytic enzymes. In C. difficile, MAL production depends on the CwlD amidase and its binding partner, the GerS lipoprotein. To gain insight into how GerS regulates CwlD activity, we solved the crystal structure of the CwlD:GerS complex. In this structure, a GerS homodimer is bound to two CwlD monomers such that the CwlD active sites are exposed. Although CwlD structurally resembles amidase_3 family members, we found that CwlD does not bind Zn2+ stably on its own, unlike previously characterized amidase_3 enzymes. Instead, GerS binding to CwlD promotes CwlD binding to Zn2+, which is required for its catalytic mechanism. Thus, in determining the first structure of an amidase bound to its regulator, we reveal stabilization of Zn2+ co-factor binding as a novel mechanism for regulating bacterial amidase activity. Our results further suggest that allosteric regulation by binding partners may be a more widespread mode for regulating bacterial amidase activity than previously thought.
UR - http://www.scopus.com/inward/record.url?scp=85118097409&partnerID=8YFLogxK
U2 - 10.1371/JOURNAL.PGEN.1009791
DO - 10.1371/JOURNAL.PGEN.1009791
M3 - Article
C2 - 34570752
AN - SCOPUS:85118097409
SN - 1553-7390
VL - 17
JO - PLoS Genetics
JF - PLoS Genetics
IS - 9
M1 - e1009791
ER -